US11519859B2

US11519859B2 - Rhodol-based thallium sensors for high-throughput screening of potassium channels

Info

Publication number: US11519859B2
Application number: US16/379,100
Authority: US
Inventors: Charles David Weaver; Brendan F. Dutter; Gary A. Sulikowski
Original assignee: Vanderbilt University
Current assignee: Vanderbilt University
Priority date: 2018-04-09
Filing date: 2019-04-09
Publication date: 2022-12-06
Also published as: US20190308991A1

Abstract

Provided are novel rhodol-based compounds, the preparation method thereof, and use thereof in Tl⁺ flux assays. The fluorescent thallium ion sensor compounds have a rhodol fluorophore attached to an amino dicarboxylic acid metal binding moiety. The compounds are prepared in the “pro-dye” form, with the rhodol oxygen and the carboxylic acids of the metal binding unit masked by protecting groups. The disclosed compounds may be used as more red-shifted and less pH-sensitive variants of Thallos.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 62/655,184, filed Apr. 9, 2018, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to novel rhodol-based compounds and their use in measuring the activities of ion channels in a cell.

BACKGROUND

Ion channels are pore-forming membrane proteins which promote the passage of ions across cellular membranes. Within the superfamily of ion channels are subfamilies with different ionic selectivity including those selective for sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺), and others. The largest subclass is K⁺ channels, which are expressed in every tissue in the body where they participate in control of excitability of neurons, muscles, endocrine glands, and the heart while in organs like the kidney they play critical roles in the formation of urine and maintenance of proper Na⁺/K⁺ homeostasis. K⁺ channel activity can be modulated by changes in transmembrane potential as well as a wide variety of extracellular and intracellular factors including but not limited to: Ca²⁺, membrane lipids, G-protein-coupled receptors, protein kinases, and ATP. A rapidly expanding number of mutations are being identified in K⁺ channels which can result in neurological, cardiovascular, renal, neoplastic, and metabolic diseases. Although K⁺ channels have been the target of therapeutic agents, the vast majority of K⁺ channels do not have potent and selective pharmacological modulators to advance the understanding of their physiological roles or to provide a foundation for developing K⁺ channel-targeted medicines. Thus, there is a desperate need to discover and characterize new K⁺ channel modulators, as well as robust chemical tools to study the K⁺ channels.

High-throughput screening (HTS) is an important method for the discovery of K⁺ channel modulators by enabling the rapid testing of thousands-to-millions of molecules to identify those with activity against the channel of interest. Success in screening depends on the development of robust, HTS-compatible assays capable of detecting subtle changes in channel activity. Electrophysiology is the most sensitive technique for assaying ion channel activity, though HTS-compatible electrophysiology technologies currently suffers from some drawbacks including high cost and lack of versatility, such as its requirement to dissociate cells from their growth substrate. An alternate approach is the use of ion flux assays where channel activity is monitored by the increase or decrease in fluorescence of an ion-sensing dye as a measure of passage of that ion through the channel of interest. In particular, the thallium (Tl⁺) flux assay is highly compatible with kinetic imaging-based HTS assays. In addition, Tl⁺ flux assays are >5-fold cheaper per well than HTS-compatible electrophysiology assays (largely due to plate cost). Tl⁺ flux has been used in numerous HTS campaigns to identify modulators of K⁺ channels implicated in disease and have also been used to screen compounds for activity against K⁺ transporters, Na⁺ channels, and non-selective cation channels.

Tl⁺ flux assays utilize the ability of Tl⁺ to pass through numerous K⁺ channels coupled with a cell-permeable fluorescent Tl⁺ indicator (a representative scheme is shown FIG. 1 ). Several fluorescent Tl⁺ indicators have been reported with the most robust, such as Thallos (1), consisting of a fluorescein moiety coupled to an amino dicarboxylic acid metal binding unit. The fluorescence pathway is partially quenched in the non-Tl⁺ bound form by a presumed photoinduced electron transfer (PET) between the metal-binding unit and fluorophore. When Tl⁺ is bound, the PET-pathway is inhibited resulting in a higher fluorescence quantum yield for the complex which is observed as a Tl⁺ concentration dependent increase in fluorescence.

Fluorescein-based metal sensors have found great utility in biology as imaging agents and components of activity assays owing to their high molar absorptivity, large fluorescence quantum yield, and general biocompatibility. However, there are drawbacks to their use including relatively short excitation wavelengths and an undesirable pH sensitivity in the physiological range leading to decreased brightness at lower pH. The short excitation wavelength is particularly a problem in HTS assays since many compound libraries contain molecules with comparable excitation and emission spectra which interfere with the assay. The number of compounds in a library generating this kind of interference tends to decrease as the excitation wavelength increases. Rhodamines, which are red-shifted ˜50 nm relative to fluorescein and exhibit excellent pH tolerance, have also been used as the fluorescent components of metal sensors, but have also been shown to accumulate in the mitochondria which is generally undesirable in flux assays. Rhodols are a hybrid of fluorescein and rhodamines exhibiting excitation and emission spectra ˜30 nm red-shifted from fluorescein, excellent pH tolerance in the physiological range, and predominantly cytoplasmic accumulation. Rhodol-based “turn-on” metal sensors have been reported for several transition metals including Hg²⁺, Ag⁺, Cu⁺ and Au³⁺. PET-based metal sensors utilizing rhodol fluorophores have been reported for imaging Cu⁺ and Ca²⁺.

There remains a need for novel metal ion sensing compounds for use in HTS assays of K⁺ channel and other Tl⁺ conducting systems, particularly those with more red-shifted fluorescence property and/or less pH-sensitivity as compared to the known compounds.

SUMMARY

The present invention provides compounds of formula (I), or a salt thereof,

wherein:

Z is O, CR^xR^y, SiR^xR^y, or SO₂;

R¹is C_1-10alkyl-CO—;

R²and R³are each independently hydrogen, C_1-10alkyl, C_1-10alkyl-CO—, —C_1-4alkylene-C_3-10cycloalkyl, C_3-10cycloalkyl, —C_1-4alkylene-C_6-20aryl, or C_6-20aryl, wherein the C_1-10alkyl; C_1-10alkyl-CO—; —C_1-4alkylene-C_3-10cycloalkyl, C_3-10cycloalkyl, —C_1-4alkylene-C_6-20aryl, and C_6-20aryl are optionally substituted with one or more substituents independently selected from the group consisting of halogen, C_1-4haloalkyl, —CN, C_1-4alkyl-O—, C_1-4alkyl-CO—NH—, and C_1-4alkyl-NH—CO—; or

R²and R³together with the N atom they are attached to form a 4 to 8-membered heterocycle, wherein the heterocycle is optionally substituted one or more substituents independently selected from the group consisting of halogen, C_1-4haloalkyl, —CN, C_1-4alkyl-O—, C_1-4alkyl-CO—NH—, and C_1-4alkyl-NH—CO—;

R⁴is C_1-10alkyl;

R⁵and R⁶are each independently C_1-6alkyl, —C_1-4-alkylene-O—R^z, —C_1-4-alkylene-O—CO—R^z, or —C_1-4-alkylene-O—C(O)—O—R^z;

R^A, R^B, R^C, and R^Dare each independently selected from the group consisting of halogen, C_1-4haloalkyl, —CN, C_1-4alkyl-, C_1-4alkyl-O—, C_1-4alkyl-CO—NH—, and C_1-4alkyl-NH—CO—;

p1, p2, p3, and p4 are each independently 0, 1, 2, or 3;

R^xand R^yare each independently C_1-4alkyl; and

R^z, at each occurrence, is independently C_1-6alkyl, C_3-10cycloalkyl, C_6-20aryl, or a 4 to 8-membered heterocycle, wherein the C_3-10cycloalkyl, C_6-20aryl, and 4 to 8-membered heterocycle are optionally substituted with one or more substituents independently selected from the group consisting of halogen, C_1-4haloalkyl, —CN, C_1-4alkyl-O—, C_1-4alkyl-CO—NH—, and C_1-4alkyl-NH—CO—.

Also provided are methods for preparing compounds of formula (I).

Also provided are methods of using compounds of formula (I) for detecting of the flux of a metal ion, such as Tl⁺, into a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an overview of Tl⁺ flux assay.

FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F show cellular localization of Thallos (FIGS. 2A, 2C), 8c (FIGS. 2B, 2D), and Rhodamine 123 (FIGS. 2C, 2F). Fluorescence microscopy images were obtained in HEK-293 cells co-expressing GIRK1 and GIRK2 following incubation with Thallos (1 μM), 8c (5 μM), or Rhodamine-123 (1 μM) for 1 h and subsequent counterstaining with Hoechst 33342 (1 μg/mL) (FIGS. 2D-F). Cells were optionally further supplemented with 1 mM of Allura Red AC before imaging (FIGS. 2A-C). Scale bar=50 μm (FIGS. 2A-C) or 10 μm (FIGS. 2D-F). Images were obtained using either a Molecular Devices ImageXpress Micro XL (FIGS. 2A-C) or a Zeiss LSM880 AiryScan with a 40×/1.30 C Plan-Apochromat Oil objective on a 8-well IbiTreat μ-Slide (Ibidi, Madison, Wis.) (FIGS. 2D-F).

FIG. 3A and FIG. 3B show the concentration response data of ML297 in HEK-293 cells co-expressing GIRK1 and GIRK2 obtained with 8c. FIG. 3A shows fluorescence intensity data from each well were normalized to the averaged fluorescence intensity of the first 6 time points of the experiment and the normalized fluorescence data for each replicate time point were averaged (n=12). FIG. 3B shows the concentration response curve derived from data in (a) by subtracting the vehicle control from each concentration series and sampling a time point several seconds after addition of Tl⁺ stimulus.

FIG. 4 shows the comparison of pH sensitivity between Thallos and 8c.

FIG. 5 shows the comparison of dynamic range between Thallos, 8c, and FluxOR Red under the indicated conditions. Tl⁺ flux assays were conducted in HEK-293 cells co-expressing GIRK1 and GIRK2 and treated with varying concentrations of the GIRK activator ML297. Fluorescence intensity data from each well were normalized to the averaged fluorescence intensity of the first 6 time points of the experiment and the normalized fluorescence data for each replicate time point were averaged (n=6-12). The plots show normalized fluorescence intensity beginning at the moment of Tl⁺ stimulus addition.

FIG. 6A and FIG. 6B show the comparison of data obtained during a Tl⁺ flux assay to generate a ML297 concentration response curve using 8c as the indicator and 529/24 nm excitation and 565/24 nm emission filters (FIG. 6A) and 544/24 nm excitation and 593/40 nm emission filters (FIG. 6B). Fluorescence intensity data from each well were normalized to the averaged fluorescence intensity of the first 6 time points of the experiment and the normalized fluorescence data for each replicate time point were averaged (n=6-12). The plots show normalized fluorescence intensity after addition of Tl⁺ stimulus.

FIG. 7 shows representative excitation and emission spectra of the compounds disclosed herein. Spectra were recorded as described and normalized to the maximum value for each condition.

DETAILED DESCRIPTION

Thallium (Tl⁺) flux assays are high-throughput screening compatible assays used for the identification of small molecule modulators of potassium (K⁺) channel activity. The assays exploit the permeability of K⁺ channels to Tl⁺ coupled with a cell permeable, fluorescent Tl⁺ sensitive dye. Common Tl⁺ sensing dyes utilize fluorescein as the fluorophore though fluorescein exhibits certain undesirable properties in these assays including pH sensitivity and spectral overlap with some molecules found in screening libraries. To overcome these drawbacks, the replacement of fluorescein with rhodols was investigated. A library of 13 rhodol-based Tl⁺ sensors was synthesized and their properties and performance in Tl⁺ flux assays evaluated. The dimethyl rhodol Tl⁺ sensor emerged as the best of the series and performed comparably to fluorescein-based sensors while demonstrating greater pH tolerance in the physiological range and excitation and emission spectra 30 nm red-shifted from fluorescein.

1. Definitions

As described herein, compounds of the invention can optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the invention. As described herein, the variables in formula I encompass specific groups, such as, for example, alkyl and cycloalkyl. As one of ordinary skill in the art will recognize, combinations of substituents envisioned by this invention are those combinations that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and preferably their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that is not substantially altered when kept at a temperature of 40° C. or less, in the absence of moisture or other chemically reactive conditions, for at least a week.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5^thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3^rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

The term “alkyl” as used herein, means a straight or branched chain saturated hydrocarbon. Representative examples of alkyl include, but are not limited to, methyl, ethyl, npropyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

The term “alkylene,” as used herein, means a divalent group derived from a straight or branched chain saturated hydrocarbon. Representative examples of alkylene include, but are not limited to, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH(CH₃)CH₂—, and CH₂CH(CH₃)CH(CH₃)CH₂—.

The term “aryl,” as used herein, means phenyl or a bicyclic aryl. The bicyclic aryl is naphthyl, dihydronaphthalenyl, tetrahydronaphthalenyl, indanyl, or indenyl. The phenyl and bicyclic aryls are attached to the parent molecular moiety through any carbon atom contained within the phenyl or bicyclic aryl.

The term “cycloalkyl” as used herein, means a monovalent group derived from an all-carbon ring system containing zero heteroatoms as ring atoms, and zero double bonds. The all-carbon ring system can be a monocyclic, bicyclic, or tricyclic ring system, and can be a fused ring system, a bridged ring system, or a spiro ring system, or combinations thereof. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The cycloalkyl groups described herein can be appended to the parent molecular moiety through any substitutable carbon atom.

The term “halogen” means a chlorine, bromine, iodine, or fluorine atom.

The term “haloalkyl,” as used herein, means an alkyl, as defined herein, in which one, two, three, four, five, six, or seven hydrogen atoms are replaced by halogen. For example, representative examples of haloalkyl include, but are not limited to, 2-fluoroethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-1, 1-dimethylethyl, and the like.

The terms “heterocycle” or “heterocyclic” refer generally to ring systems containing at least one heteroatom as a ring atom where the heteroatom is selected from oxygen, nitrogen, and sulfur. In some embodiments, a nitrogen or sulfur atom of the heterocycle is optionally substituted with oxo. Heterocycles may be a monocyclic, bicyclic, or tricyclic ring system, and can be a fused ring system, a bridged ring system, or a spiro ring system, or combinations thereof. The monocyclic heterocycle is generally a 4, 5, 6, 7, or 8-membered non-aromatic ring containing at least one heteroatom selected from O, N, or S. The 4-membered ring contains one heteroatom and optionally one double bond. The 5-membered ring contains zero or one double bond and one, two or three heteroatoms. The 6, 7, or 8-membered ring contains zero, one, or two double bonds, and one, two, or three heteroatoms. Representative examples of monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, diazepanyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl, 4,5-dihydroisoxazol-5-yl, 3,4-dihydropyranyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl, thiopyranyl, and trithianyl. The fused bicyclic heterocycle is a 7-12-membered ring system having a monocyclic heterocycle fused to a phenyl, to a saturated or partially saturated carbocyclic ring, or to another monocyclic heterocyclic ring, or to a monocyclic heteroaryl ring. Representative examples of fused bicyclic heterocycle include, but are not limited to, 1,3-benzodioxol-4-yl, 1,3-benzodithiolyl, 3-azabicyclo[3.1.0]hexanyl, hexahydro-1H-furo[3,4-c]pyrrolyl, 2,3-dihydro-1,4-benzodioxinyl, 2,3-dihydro-1-benzofuranyl, 2,3-dihydro-1-benzothienyl, 2,3-dihydro-1H-indolyl, 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazinyl, and 1,2,3,4-tetrahydroquinolinyl. Spiro heterocycle means a 4-, 5-, 6-, 7-, or 8-membered monocyclic heterocycle ring wherein two of the substituents on the same carbon atom form a second ring having 3, 4, 5, 6, 7, or 8 members. Examples of a spiro heterocycle include, but are not limited to, 1,4-dioxa-8-azaspiro[4.5]decanyl, 2-oxa-7-azaspiro[3.5]nonanyl, 2-oxa-6-azaspiro[3.3]heptanyl, and 8-azaspiro[4.5]decane. The monocyclic heterocycle groups of the present invention may contain an alkylene bridge of 1, 2, or 3 carbon atoms, linking two nonadjacent atoms of the group. Examples of such a bridged heterocycle include, but are not limited to, 2,5-diazabicyclo[2.2.1]heptanyl, 2-azabicyclo[2.2.1]heptanyl, 2-azabicyclo[2.2.2]octanyl, and oxabicyclo[2.2.1]heptanyl. The monocyclic, fused bicyclic, and spiro heterocycle groups are connected to the parent molecular moiety through any substitutable carbon atom or any substitutable nitrogen atom contained within the group.

The term “oxo” as used herein refers to an oxygen atom bonded to the parent molecular moiety. An oxo may be attached to a carbon atom or a sulfur atom by a double bond. Alternatively, an oxo may be attached to a nitrogen atom by a single bond, i.e., an N-oxide.

Terms such as “alkyl,” “cycloalkyl,” “alkylene,” etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance (e.g., “C_1-4alkyl,” “C_3-6cycloalkyl,” “C_1-4alkylene”). These designations are used as generally understood by those skilled in the art. For example, the representation “C” followed by a subscripted number indicates the number of carbon atoms present in the group that follows. Thus, “C₃alkyl” is an alkyl group with three carbon atoms (i.e., n-propyl, isopropyl). Where a range is given, as in “C_1-4,” the members of the group that follows may have any number of carbon atoms falling within the recited range. A “C_1-4alkyl,” for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).

Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Thus, included within the scope of the invention are tautomers of compounds of formula I. The structures also include zwitterioinc forms of the compounds or salts of formula I where appropriate.

2. Compounds

A first aspect of the invention provides compounds of formula (I), or a salt thereof,

wherein:

Z is O, CR^xR^y, SiR^xR^y, or SO₂;

R¹is C_1-10alkyl-CO—;

R⁴is C_1-10alkyl;

p1, p2, p3, and p4 are each independently 0, 1, 2, or 3;

R^xand R^yare each independently C_1-4alkyl; and

In some embodiments, Z is O. In some embodiments, Z is CR^xR^y, SiR^xR^y, or SO₂. In some embodiments, Z is C(CH₃)₂, Si(CH₃)₂, or SO₂.

In some embodiments, p1, p2, p3, and p4 are each independently 0 or 1. In some embodiments, p1, p2, p3, and p4 are 0.

In some embodiments, R^A, R^B, R^C, and R^Dare each independently halogen, C_1-4haloalkyl, or —CN. In some embodiments, R^A, R^B, R^C, and R^Dare each independently a halogen atom. In some embodiments, R^Ais halogen, and p1 is 1. In some embodiments, R^Bis halogen, and p2 is 1. In some embodiments, R^Cis halogen, and p3 is 1. In some embodiments, R^Dis halogen, and p4 is 1.

In some embodiments, R¹is C_1-4alkyl-CO—. In some embodiments, R¹is CH₃—CO—.

In some embodiments, R²and R³are each independently hydrogen, C_1-10alkyl, C_1-10alkyl-CO—, —C_1-4alkylene-C_3-10cycloalkyl, C_3-10cycloalkyl, —C_1-4alkylene-C_6-20aryl, or C_6-20aryl, wherein the C_1-10alkyl, C_1-10alkyl-CO—, —C_1-4alkylene-C_3-10cycloalkyl, C_3-10cycloalkyl, —C_1-4alkylene-C_6-20aryl, and C_6-20aryl are optionally substituted with one or more substituents independently selected from the group consisting of halogen, C_1-4haloalkyl, —CN, C_1-4alkyl-O—, C_1-4alkyl-CO—NH—, and C_1-4alkyl-NH—CO—.

In some embodiments, R²and R³are each independently hydrogen, C_1-4alkyl, or C_3-10cycloalkyl (such as cyclohexyl). In some embodiments, R²and R³are both hydrogen. In some embodiments, R²is hydrogen, and R³is methyl, ethyl, propyl, or butyl. In some embodiments, R²and R³are each independently C_1-4alkyl. In some embodiments, R²and R³are both methyl, ethyl, or propyl.

In some embodiments, R²is hydrogen, and R³is C_3-10cycloalkyl. For example, R³may be a monocyclic C_3-10cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, R²is hydrogen, and R³is cyclohexyl.

In some embodiments, R²and R³together with the N atom they are attached to form a 4 to 8-membered heterocycle, wherein the heterocycle is optionally substituted one or more substituents independently selected from the group consisting of halogen, C_1-4haloalkyl, —CN, C_1-4alkyl-O—, C_1-4alkyl-CO—NH—, and C_1-4alkyl-NH—CO—. For example, in some embodiments, the heterocycle is

In some embodiments, the heterocycle is not substituted. In some embodiments, the heterocycle is substituted with one or more substituents independently selected from the group consisting of halogen, C_1-4haloalkyl, —CN, C_1-4alkyl-O—, C_1-4alkyl-CO—NH—, and C_1-4alkyl-NH—CO—. In some embodiments, the heterocycle is substituted with 1, 2, 3, 4, or 5 halogen groups. For example, in some embodiments, the heterocycle is

In some embodiments, R²and R³together with the N atom they are attached to form a 4 to 8-membered heterocycle, wherein the heterocycle contains at least one heteroatom in addition to the N atom to which R²and R³are attached. For example, in some embodiments, the heterocycle is

In some embodiments, R⁴is C_1-4alkyl. In some embodiments, R⁴is methyl.

In general, R⁵and R⁶may be cleaved by an esterase to convert the —OR⁵and —OR⁶groups to —OH. In some embodiments, R⁵and R⁶have the same structure. In some embodiments, R⁵and R⁶are independently C_1-6alkyl, such as methyl, ethyl, propyl, isopropyl, or t-butyl.

In some embodiments, R⁵and R⁶are independently —C_1-4-alkylene-O—R^z, —C_1-4-alkylene-O—CO—R^z, or —C_1-4-alkylene-O—C(O)—O—R^z. In some embodiments, R⁵and R⁶are independently —CH₂—O—R^z, —CH₂—O—CO—R^z, or —CH₂—O—C(O)—O—R^z. In some embodiments, R⁵and R⁶are independently —CH₂—O—CO—R^z.

In some embodiments, each R^zis independently a C_1-6alkyl, such as methyl, ethyl, propyl, isopropyl, or t-butyl. In some embodiments, R⁵and R⁶are —CH₂—O—CO—R^z, wherein R^xis C_1-6alkyl. In some embodiments, R⁵and R⁶are —CH₂—O—CO—CH₃.

In some embodiments, each R^zis independently C_3-10cycloalkyl, C_6-20aryl, or 4 to 8-membered heterocycle, wherein the C_3-10cycloalkyl, C_6-20aryl, and 4 to 8-membered heterocycle are optionally substituted with one or more substituents independently selected from the group consisting of halogen, C_1-4haloalkyl, —CN, C_1-4alkyl-O—, C_1-4alkyl-CO—NH—, and C_1-4alkyl-NH—CO—. In some embodiments, each R^zis independently a C_3-10cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, which is unsubstituted or substituted with 1, 2, 3, or 4 substituents independently selected from the group consisting of halogen, C_1-4haloalkyl, —CN, C_1-4alkyl-O—, C_1-4alkyl-CO—NH—, and C_1-4alkyl-NH—CO—. In some embodiments, each R^zis independently a C_6-20aryl, such as phenyl, which is unsubstituted or substituted with 1, 2, 3, or 4 substituents selected from the group consisting of halogen, C_1-4haloalkyl, —CN, C_1-4alkyl-O—, C_1-4alkyl-CO—NH—, and C_1-4alkyl-NH—CO—.

In some embodiments, the compounds disclosed herein have a structure of formula (I-a), or a salt thereof,

wherein R², R³, R⁵, and R⁶are as described herein.

In some embodiments, the compound of formula (I) is selected from

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-amino-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetamido-6′-acetoxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(diethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dipropylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dibutylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(cyclohexylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(butylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-3-oxo-6′-(phenylamino)-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(3,3-difluoroazetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-3-oxo-6′-(pyrrolidin-1-yl)-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate; and
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-3-oxo-6′-(piperidin-1-yl)-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate,
or a salt thereof.

In another embodiment, the compounds include isotope-labelled forms. An isotope-labelled form of a compound is identical to the compound apart from the fact that one or more atoms of the compound have been replaced by an atom or atoms having an atomic mass or mass number which differs from the atomic mass or mass number of the atom which usually occurs in greater natural abundance. Examples of isotopes which are readily commercially available and which can be incorporated into a compound by well-known methods include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, for example ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ¹⁸F and ³⁶Cl.

3. Chemical Synthesis

In another aspect, provided are methods of preparing of compounds of formula (I), or salts thereof. In some embodiments, the method comprises reacting a compound of formula (II)

with a compound of formula (III)

to the compound of formula (I), wherein Z, R¹, R², R³, R⁴, R⁵, R⁶, R^A, R^B, R^C, R^D, p1, p2, p3, and p4 are as described herein.

In some embodiments, the method further comprises converting a compound of formula (IV)

to the compound of formula (II), wherein Z, R^A, R^B, R^C, p1, p2, and p3, are as described herein.

In some embodiments, a variable route to the rhodol component of the Tl⁺ sensor was envisioned utilizing a similar strategy as previously described but incorporating protecting groups to allow for orthogonal deprotection of the phenol and distal carboxylic acid functional groups. Scheme 1 shows a representative synthesis route for compounds of formula (I), wherein Z is O.

For example, starting from 5-carboxyfluorescein (2), mono-phenol protection as the benzyl ether was accomplished by treatment with benzyl chloride and potassium carbonate in DMF under microwave heating followed by benzyl ester saponification with lithium hydroxide to afford 3. Carboxylic acid 3 was then protected as the t-butyl ester and converted to triflate 4. From this common intermediate, rhodols 5a-m were prepared by Buchwald-Hartwig coupling with the corresponding amine. These compounds were converted to O-acetyl rhodols 6a-m by sequential hydrogenolysis of the benzyl ether, removal of the t-butyl ester by treatment with TFA, and O-acetylation, with the exceptions of 6a, which was O-acetylated prior to t-butyl ester removal and N-Boc deprotection and 6b which was O- and N-acetylated following treatment with TFA. Carboxylic acids 6a-m were coupled to 7 to provide rhodol Tl⁺ sensors 8a-m (Table 1).

TABLE 1

Photochemical properties of rhodol Tl⁺ sensors 8a-m. ^{[a, b]}

Com-					on/off (Tl⁺ flux
pound	X	λ_max	λ_em	ε (M⁻¹ * cm⁻¹)	assay)

8a		496	520	57000	3.02 ± 0.08

8b		463	NA	24000	NA

8c		521	551	56000	4.21 ± 0.04

8d		525	551	55000	3.28 ± 0.04

8e		527	553	66000	2.94 ± 0.08

8f		528	556	58000	1.52 ± 0.01

8g		514	535	57000	4.26 ± 0.07

8h		511	533	48000	3.50 ± 0.07

8i		517	NA	52000	1.07 ± 0.01

8j		522	550	56000	3.92 ± 0.13

8k		506	530	66000	2.02 ± 0.04

8l		525	553	39000	3.54 ± 0.07

8m		526	557	48000	3.09 ± 0.04

^[a] Absorption maxima, fluorescence emission maxima (both in nm), and molar absorptivity were measured after saponification of 8a-m and dilution into 150 mM KCl buffered with 10 mM HEPES and supplemented with 50 μM EDTA at pH = 7.22.
^[b] In cell on/off ratios were determined using HEK-293 cells co-expressing GIRK1 and GIRK2 treated with 8a-m for 1 h and then subjected to Tl⁺ stimulus. Values represent the maximum fold increase in fluorescence after Tl⁺ addition (n = 6).

4. Methods of Use

Disclosed are fluorescent thallium ion sensor compounds having a rhodol fluorophore attached to an amino dicarboxylic acid metal binding moiety. These compounds are also referred to herein as metal sensors or dye molecules. These dye molecules are prepared in the “pro-dye” form, with the rhodol oxygen and the carboxylic acids of the metal binding unit masked by protecting groups. These modifications allow for better cell uptake. The protecting groups may be cleaved by cytoplasmic esterase resulting in the active dye molecules, which produce fluorescence signal upon complexing with the metal ion, such as thallium ion (Tl⁺). The active dye molecules may yield higher fluorescence signal when complexed with a metal ion (i.e. the “metal-bound” state) than that in the absence of the metal ion (i.e. the “unbound” state). Due to their photophysical properties, the compounds disclosed herein are useful in in vitro and cellular assays for measuring metal flux, such as the flux of thallium ion (Tl⁺). For example, such measurement may be made based on the initial brightness produced by the active compounds in the absence of the metal ion, and the fold-increase in brightness in the presence of the metal ion.

In one aspect, provided is a method of detecting flux of a metal ion into a cell, the method comprising

(a) contacting the cell with a compound as disclosed herein, or a salt thereof;

(b) subsequently contacting the cell with the metal ion; and

(c) subsequently measuring the fluorescence produced by the cell.

In some embodiments, the metal ion is Tl⁺.

Suitable cells as disclosed herein include those known in the art for cellular studies of ion channels, such as HEK-293 cells. In some embodiments, the cell comprises at least one ion channel, such as sodium (Na⁺), potassium (K⁺), and calcium (Ca²⁺) and others. In some embodiments, the ion channel comprises a potassium channel, such as a G-protein gated inward rectifying K⁺ channel (GIRK). In some embodiments, the cell may comprise multiple ion channels. In some embodiments, the method as disclosed herein is carried out in HEK-293 cells co-expressing multiple ion channels, such as GIRK1 and GIRK2.

In some embodiments, the method may further comprise measuring the fluorescence produced by the cell after step (a) and before step (b). For example, the method may comprise measuring the initial fluorescence after contacting a compound disclosed herein with a cell in the absence of the metal ion, and the fluorescence of the cell in the presence of the metal ion. In some embodiments, after contacting a compound disclosed herein with a cell, a ratio (e.g. fold increase) between the fluorescence produced by the cell in the absence of a metal ion and that in the presence of the metal ion may be calculated.

5. Example

Method and Materials

General Chemical Synthesis Methods

All non-aqueous reactions were performed in flame-dried flasks under an atmosphere of argon. Stainless steel syringes were used to transfer air- and moisture-sensitive liquids. Reaction temperatures were controlled using a thermocouple thermometer and analog hotplate stirrer. Reactions were conducted at room temperature (rt, approximately 23° C.) unless otherwise noted. Flash column chromatography was conducted using silica gel 230-400 mesh. Analytical thin-layer chromatography (TLC) was performed on E. Merck silica gel 60 F254 plates and visualized using UV and iodine stain.

Materials

Acetic anhydride, trifluoromethanesulfonic anhydride, and HATU were purchased from Oakwood Products (Estill, S.C.). Diisopropylamine, azetidine hydrochloride, 3,3-di-fluoroazetidine hydrochloride, and cesium carbonate were purchased from Combi-Blocks (San Diego, Calif.). Pyrrolidine was purchase from Acros. 5-carboxyfluorescein (Hammershøj et al., European J. Org. Chem. 2015, 2015, 7301-7309) and O-tert-butyl-di-isopropylisourea (West et al., Org. Lett. 2005, 7, 2615-2618) were prepared according to literature procedures. All other solvents and chemicals were purchased from Sigma-Aldrich N,N-dimethylformamide (DMF), dichloromethane, pyridine, toluene (PhMe), triethylamine (Et₃N), and dimethylsulfoxide (DMSO) were used as received in a bottle with a Sure/Seal. Trifluoromethanesulfonic anhydride was distilled from P₂O₅prior to use. Deuterated solvents were purchased from Cambridge Isotope Laboratories.

Instrumentation

¹H NMR spectra were recorded on Bruker 400 or 600 MHz spectrometers and are reported relative to deuterated solvent signals. Data for ¹H NMR spectra are reported as follows: chemical shift (δ ppm), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, p=pentet, m=multiplet, br=broad, app=apparent), coupling constants (Hz), and integration. ¹³C NMR spectra were recorded on Bruker 100 or 150 MHz spectrometers and are reported relative to deuterated solvent signals. Low resolution mass spectrometry (LRMS) was conducted and recorded on an Agilent Technologies 6130 Quadrupole instrument. Preparative scale HPLC was conducted on a Gilson HPLC machine. Automated flash chromatography was performed on a Teledyne Isco purification system.

Example 1 Compound Synthesis

Synthetic Procedures and Compound Characterization

3′-(benzyloxy)-6′-hydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxylic acid (3)

To a stirred solution of 1.35 g (3.59 mmol, 1.0 eq) of 2 in 10 mL of DMF in a 20 mL microwave vial was added 2.48 g (18.0 mmol, 5.0 eq) of K₂CO₃and 1.66 mL (14.4 mmol, 4.0 eq) of benzyl chloride. The vial was capped, flushed with Ar, and maintained at 120° C. under microwave irradiation for 1 h. The reaction was poured into 1 N HCl, the mixture extracted with EtOAc (3×), the organic layers combined and washed with brine, and concentrated. The residue was dissolved in 120 mL of THF and 12 mL of H₂O. To this solution was added 431 mg (18.0 mmol, 5.0 eq) of LiOH. The solution was refluxed for 1 h when the reaction was deemed complete by LCMS. The solution was acidified with 1 N HCl and THE removed in vacuo. The resulting mixture was extracted with ethyl acetate (3×), the organic layers were combined, washed with brine, and passed through a phase separator (Biotage). The solution was concentrated and the residue was purified by flash chromatography with hexane and ethyl acetate plus 1% acetic acid to provide 0.99 g (59%) of 3. ¹H NMR (400 MHz, acetone-d6) δ 8.56-8.55 (app q, 1H), 8.40 (dd, J=8.02 Hz, J=1.50 Hz, 1H), 7.51-7.47 (m, 2H), 7.43-7.36 (m, 3H), 7.35-7.30 (m, 1H), 6.96-6.95 (app d, 1H), 6.83-6.77 (m, 3H), 6.74 (d, J=8.64 Hz, 1H), 6.65 (dd, J=8.68 Hz, J=2.40 Hz, 1H), 5.20 (s, 2H); ¹³C NMR (100 MHz, acetone-d6) δ 168.6, 166.3, 161.6, 160.5, 157.6, 153.2, 137.7, 137.0, 133.5, 130.2, 130.1, 129.4, 128.8, 128.5, 128.3, 126.8, 125.4, 113.6, 113.4, 112.0, 110.8, 103.4, 102.6, 83.8, 70.8; LRMS calculated for C₂₈H₁₈O₇[M+H]⁺ m/z: 467.1, measured 467.0.

tert-butyl 3′-(benzyloxy)-6′-hydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxylate (S1)

To a stirred solution of 0.49 g (1.05 mmol, 1.0 eq) of 3 in pyridine (5 mL) was added 149 μL (1.58 mmol, 1.5 eq) of acetic anhydride. The solution was stirred at room temperature for 2 h until judged complete by LCMS. The solution was concentrated, the resulting residue acidified with 1 N HCl, extracted with ethyl acetate (3×), washed with brine, and passed through a phase separator (Biotage). The solution was concentrated in vacuo and the residue was dissolved in dichloromethane (5 mL). To this solution was added 630 mg (3.15 mmol, 3.0 eq) of O-t-Bu-DIIU. The solution was stirred for 48 h at room temperature. The resulting suspension was filtered, the solids washed with dichloromethane, the organics combined and concentrated, and the residue dissolved in methanol (5 mL). To this solution was added 1.1 mL (5.25 mmol, 5.0 eq) of 25% sodium methoxide in methanol. The mixture was stirred for 20 min at room temperature, quenched with 1 N HCl, concentrated and extracted with ethyl acetate (3×). The organic layers were combined, washed with brine, passed through a phase separator (Biotage), and concentrated in vacuo. The residue was purified by flash chromatography to provide 0.37 g (68%) of S1. ¹H NMR (400 MHz, CDCl₃) δ 8.62 (s, 1H), 8.29 (dd, J=8.02 Hz, J=1.34 Hz, 1H), 7.44-7.30 (m, 5H), 7.21 (d, J=8.04 Hz, 1H), 6.83 (d, J=2.08 Hz, 1H), 6.74 (d, J=2.00 Hz, 1H), 6.70-6.62 (m, 2H), 6.57-6.50 (m, 2H), 5.07 (s, 2H), 1.64 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 169.5, 164.4, 160.8, 158.6, 156.6, 152.6, 136.3, 136.2, 134.2, 129.2, 129.1, 128.8, 127.6, 127.1, 126.6, 124.3, 112.7, 112.6, 110.7, 110.3, 103.4, 102.1, 84.7, 82.7, 70.4, 28.3; LRMS calculated for C₃₂H₂₆O₇[M+H]⁺ m/z: 523.2, measured 523.6.

tert-butyl 3′-(benzyloxy)-3-oxo-6′-(((trifluoromethyl)sulfonyl)oxy)-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxylate (4)

To a stirred solution of 0.37 g (0.709 mmol, 1.0 eq) of S1 and 228 μL (2.84 mmol, 4.0 eq) of pyridine in dichloromethane (5 mL) at 0° C. was added 238 μL (1.42 mmol, 2.0 eq) of trifluoromethanesulfonic anhydride. The solution was stirred at 0° C. for 30 min at which point the reaction was judged complete by TLC. The solution was concentrated and the resulting residue purified by flash chromatography to provide 0.36 g (78%) of 4 as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.63 (d, J=0.60 Hz, 1H), 8.32 (dd, J=8.02 Hz, J=1.42 Hz, 1H), 7.44-7.32 (m, 5H), 7.27-7.25 (m, 1H), 7.22 (d, J=7.88 Hz, 1H), 6.96 (dd, J=8.80 Hz, J=2.40 Hz, 1H), 6.90-6.86 (m, 2H), 6.74 (dd, J=8.84 Hz, J=2.36 Hz, 1H), 6.69 (d, J=8.80 Hz, 1H), 5.10 (s, 2H), 1.64 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 168.3, 164.0, 161.0, 156.0, 152.1, 151.9, 150.2, 136.5, 134.7, 130.1, 129.1, 128.9, 128.4, 127.6, 126.8, 126.7, 124.1, 119.3, 116.9, 113.4, 110.8, 110.3, 102.2, 82.7, 81.8, 70.5, 28.3; ¹⁹F NMR (376 MHz, CDCl₃) δ 75.7; LRMS calculated for C₃₃H₂₅F₃O₉S [M+H]+m/z: 655.1, measured 655.5.

General Procedure for Buchwald-Hartwig Coupling

To a stirred solution of triflate 4 (1.0 eq) dissolved in toluene (0.08 M) in a microwave vial was added Pd(OAc)₂(0.1 eq), R-BINAP (0.15 eq), Cs₂CO₃(4 eq), and amine (1.2 eq). The tube was sealed and maintained at 100° C. on a heating block for 18 h. The mixture was cooled, filtered through celite, and concentrated in vacuo. The crude residue was purified by flash chromatography to provide products 5a-m (18-90%).

General Hydrogenolysis Procedure

To a stirred solution of benzyl ether (1.0 eq) in ethyl acetate (1 mL) was added 10% palladium on carbon (0.05 mol % of palladium). The solution was flushed with Ar and then H₂. The reaction was vigorously stirred at room temperature under an atmosphere of H₂until it was judged complete by LCMS (˜1 h). The reaction was flushed with Ar, filtered through celite, and concentrated to provide crude phenol. The material was carried on crude to the next step.

General Procedure for t-Butyl Ester Deprotection

To a suspension of phenol (1.0 eq) in DCM (2.0 mL) was added TFA (400 μL). The resulting solution was stirred overnight at room temperature. The reaction was judged complete by LCMS and concentrated. The product was concentrated and carried on crude to the next step.

General O-Acetylation Procedure

To a solution of phenol in pyridine (1.0 mL) was added acetic anhydride (1.5 eq). The solution was stirred until judged complete by LCMS. The solution was concentrated, dissolved in DMSO, and purified by preparative scale reverse phase HPLC using an acetonitrile and water with 0.1% TFA gradient. Fractions containing product were lyophilized to provide 6a-m (22-67%).

Synthesis of 6a and 6b.

The common intermediate for the synthesis of 6a and 6b was prepared by Buchwald-Hartwig coupling of 4 with N-tert-butylcarbamate followed by hydrogenolysis of the benzyl ether as described above. 6a was prepared by O-acetylation followed by ester and N-boc deprotection under the t-butyl ester deprotection conditions described. 6b was prepared first by ester and N-boc deprotection followed by bis-acetylation accomplished by increasing the amount of acetic anhydride in the above O-acetylation conditions to 3 equivalents.

General Coupling Procedure

To a stirred solution of 6a-m in DMSO was added HATU (1.5 eq), triethylamine (2 eq), and 7 (C. D. Weaver, Thallium Fluorescent Ion Indicator and Assay, 2015, U.S. Pat. No. 9,103,791 B1) (1.1 eq). The solution was stirred until judged complete by LCMS (1-2 h). The product was purified by preparative reverse phase HPLC using an acetonitrile and water with 0.1% TFA gradient. Fractions containing product were lyophilized to provide 8a-m (33-58%).

Compound Storage

Compounds 8a-m were dissolved in DMSO and stored as 10 mM stock solutions at −20° C. These conditions were generally well tolerated with the exception of 8a which appeared to undergo transacetylation and equilibrated to a mixture of 8a, 8b, and mono-deacetylated species upon storage. To prevent this, dry aliquots of 8a were prepared and dissolved in DMSO immediately before use.

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-amino-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate (8a)

¹H NMR (600 MHz, CD₃CN) δ 8.93 (s, 1H), 8.60 (s, 1H), 8.29 (d, J=7.80 Hz, 1H), 7.44 (s, 1H), 7.40 (d, J=7.86 Hz, 1H), 7.25 (s, 1H), 7.20 (d, J=8.04 Hz, 1H), 7.01 (d, J=8.64 Hz, 1H), 6.95 (d, J=8.16 Hz, 1H), 6.83 (d, J=8.58 Hz, 1H), 6.78 (d, J=8.58 Hz, 1H), 6.65 (s, 1H), 6.59 (d, J=8.34 Hz, 1H), 5.71 (s, 4H), 4.13 (s, 4H), 3.80 (s, 3H), 2.28 (s, 3H), 2.07 (s, 6H); ¹³C NMR (150 MHz, CD₃CN) δ 171.1, 170.5, 170.0, 168.5, 164.8, 155.5, 154.9, 153.6, 152.2, 138.2, 135.9, 134.9, 134.4, 131.2, 130.5, 129.0, 127.1, 126.6, 119.9, 119.8, 115.6, 113.9, 111.5, 106.7, 100.6, 80.1, 56.3, 54.4, 21.3, 20.9; LRMS calculated for C₄₀H₃₅N₃O₁₅[M+H]⁺ m/z: 798.2; measured 798.4.

bis(acetoxymethyl) 2,2′-((4-(3′-acetamido-6′-acetoxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate (8b)

¹H NMR (600 MHz, CD₃CN) δ 8.88 (s, 1H), 8.55-8.51 (app d, 2H), 8.27 (dd, J=8.01 Hz, J=1.17 Hz, 1H), 7.82 (d, J=1.62 Hz, 1H), 7.45 (d, J=1.74 Hz, 1H), 7.37 (d, J=8.04 Hz, 1H), 7.19 (dd, J=8.58 Hz, J=2.04 Hz, 1H), 7.15 (d, J=2.16 Hz, 1H), 7.11 (dd, J=8.67 Hz, J=2.01 Hz, 1H), 6.91 (d, J=8.70 Hz, 1H), 6.86 (dd, J=8.64 Hz, J=2.16 Hz, 1H), 6.83 (d, J=8.58 Hz, 1H), 6.80 (d, J=8.64 Hz, 1H), 5.71 (s, 4H), 4.13 (s, 4H), 3.81 (s, 3H), 2.27 (s, 3H), 2.08 (s, 3H), 2.06 (s, 6H); ¹³C NMR (150 MHz, CD₃CN) δ 171.0, 170.5, 170.1, 169.2, 164.8, 156.3, 153.6, 152.7, 152.3, 152.2, 142.6, 138.4, 135.9, 134.5, 130.1, 129.5, 127.6, 125.3, 124.9, 119.9, 119.1, 117.2, 116.2, 113.84, 113.77, 111.5, 83.0, 80.1, 56.3, 54.4, 24.5, 21.2, 20.9; LRMS calculated for C₄₂H₃₇N₃O₁₆[M+H]⁺ m/z: 840.2, measured 840.3.

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxamido)-2-methoxyphenyl)azanediyl)diacetate (8c)

¹H NMR (600 MHz, acetone-d6) δ 9.76 (s, 1H), 8.55 (s, 1H), 8.40 (d, J=7.98 Hz, 1H), 7.64-7.61 (m, 1H), 7.46 (d, J=8.04 Hz, 1H), 7.38-7.34 (m, 1H), 7.16 (d, J=1.80 Hz, 1H), 6.94-6.88 (m, 2H), 6.86 (d, J=8.58 Hz, 1H), 6.67 (d, J=8.94 Hz, 1H), 6.60-6.55 (m, 2H), 5.78 (s, 4H), 4.19 (s, 4H), 3.83 (s, 3H), 3.02 (s, 6H), 2.29 (s, 3H), 2.07 (s, 6H); ¹³C NMR (150 MHz, acetone-d6) δ 170.8, 169.9, 169.4, 168.8, 164.4, 164.3, 156.1, 153.5, 153.4, 153.2, 153.0, 152.2, 138.3, 136.0, 135.5, 134.9, 134.8, 129.9, 129.5, 128.2, 125.3, 124.3, 119.8, 118.6, 117.7, 113.5, 113.4, 111.2, 110.4, 106.5, 106.4, 106.3, 99.0, 79.9, 56.2, 54.4, 40.3, 21.0, 20.6; LRMS calculated for C₄₂H₃₉N₃O₁₅[M+H]⁺ m/z: 826.3, measured 826.5.

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(diethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate (8d)

¹H NMR (600 MHz, acetone-d6) δ 9.76 (s, 1H), 8.56 (s, 1H), 8.41 (dd, J=7.95 Hz, J=0.81 Hz, 1H), 7.63 (d, J=1.98 Hz, 1H), 7.49 (d, J=8.04 Hz, 1H), 7.36 (dd, J=8.58 Hz, J=2.10 Hz, 1H), 7.18 (d, J=1.50 Hz, 1H), 6.95-6.89 (m, 2H), 6.86 (d, J=8.58 Hz, 1H), 6.68 (d, J=8.58 Hz, 1H), 6.61-6.57 (m, 2H), 5.78 (s, 4H), 4.20 (s, 4H), 3.83 (s, 3H), 3.48 (q, J=7.02 Hz, 4H), 2.29 (s, 3H), 2.07 (s, 6H), 1.19 (t, J=7.02 Hz, 6H); ¹³C NMR (150 MHz, acetone-d6) δ 170.8, 169.9, 169.3, 168.8, 164.4, 155.9, 153.6, 153.4, 153.0, 152.2, 150.9, 138.3, 136.0, 135.5, 134.9, 129.9, 129.8, 128.4, 125.4, 124.4, 119.8, 118.5, 117.8, 113.5, 111.2, 110.0, 106.5, 105.6, 87.3, 79.9, 56.2, 54.4, 45.1, 21.0, 20.6, 12.7; LRMS calculated for C₄₄H₄₃N₃O₁₅[M+H]⁺ m/z; 854.3, measured 854.5.

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dipropylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate (8e)

¹H NMR (600 MHz, acetone-d6) δ 9.75 (s, 1H), 8.54 (s, 1H), 8.40 (dd, J=7.98 Hz, J=1.14 Hz, 1H), 7.63 (d, J=2.16 Hz, 1H), 7.47 (d, J=7.98 Hz, 1H), 7.36 (dd, J=8.58 Hz, J=2.22 Hz, 1H), 7.16 (d, J=1.74 Hz, 1H), 6.93-6.88 (m, 2H), 6.86 (d, J=8.58 Hz, 1H), 6.63 (d, J=8.58 Hz, 1H), 6.54-6.51 (m, 2H), 5.78 (s, 4H), 4.20 (s, 4H), 3.83 (s, 3H), 3.36 (t, J=7.68 Hz, 4H), 2.28 (s, 3H), 2.07 (s, 6H), 1.64 (sext, J=7.48 Hz, 4H), 0.94 (t, J=7.38 Hz, 6H); ¹³C NMR (150 MHz, acetone-d6) δ 170.8, 170.0, 169.3, 168.8, 164.4, 153.5, 153.4, 153.0, 152.2, 151.3, 138.3, 136.0, 135.5, 134.9, 129.9, 129.6, 128.4, 125.4, 124.3, 119.8, 118.5, 117.8, 113.5, 111.2, 110.1, 106.5, 105.5, 98.3, 79.9, 56.2, 54.4, 53.2, 30.3, 21.0, 20.6, 11.5; LRMS calculated for C₄₆H₄₇N₃O₁₅[M+H]⁺ m/z; 882.3, measured 882.5.

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dibutylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate (8f)

¹H NMR (600 MHz, acetone-d6) δ 9.77 (s, 1H), 8.54 (s, 1H), 8.40 (d, J=7.98 Hz, 1H), 7.63 (d, J=2.10 Hz, 1H), 7.47 (d, J=7.98 Hz, 1H), 7.38-7.35 (m, 1H), 7.16 (d, J=1.50 Hz, 1H), 6.92-6.87 (m, 2H), 6.86 (d, J=8.64 Hz, 1H), 6.62 (d, J=8.64 Hz, 1H), 6.54-6.50 (m, 2H), 5.78 (s, 4H), 4.20 (s, 4H), 3.83 (s, 3H), 3.38 (t, J=7.68 Hz, 4H), 2.28 (s, 3H), 2.07 (s, 6H), 1.61 (p, J=7.64 Hz, 4H), 1.38 (sext, J=7.51 Hz, 4H), 0.95 (t, J=7.38 Hz, 6H); ¹³C NMR (150 MHz, acetone-d6) δ 170.8, 169.9, 169.3, 156.1, 153.5, 153.3, 153.0, 152.2, 151.2, 138.3, 136.0, 135.5, 134.9, 129.9, 129.6, 128.3, 125.4, 124.3, 119.8, 118.5, 117.8, 113.5, 111.2, 110.0, 106.5, 105.3, 98.3, 79.9, 56.2, 54.4, 51.2, 21.0, 20.8, 20.6, 14.2; LRMS calculated for C₄₈H₅₁N₃O₁₅[M+H]⁺ m/z: 910.3, measured 910.6.

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(cyclohexylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate (8g)

¹H NMR (600 MHz, acetone-d6) δ 9.76 (s, 1H), 8.54 (s, 1H), 8.40 (d, J=7.26 Hz, 1H), 7.62 (d, J=2.10 Hz, 1H), 7.47 (d, J=8.04 Hz, 1H), 7.36 (dd, J=8.58 Hz, J=2.22 Hz, 1H), 7.15 (s, 1H), 6.89 (s, 2H), 6.85 (d, J=8.64 Hz, 1H), 6.55 (d, J=8.70 Hz, 1H), 6.50 (d, J=2.04 Hz, 1H), 6.46 (dd, J=8.73 Hz, J=2.19 Hz, 1H), 5.78 (s, 4H), 4.19 (s, 4H), 3.83 (s, 3H), 3.40-3.43 (m, 2H), 2.28 (s, 3H), 2.07 (s, 6H), 1.80-1.74 (s, 2H), 1.67-1.62 (m, 1H), 1.48-1.39 (m, 2H), 1.31-1.19 (m, 3H); ¹³C NMR (150 MHz, acetone-d6) δ 170.8, 169.9, 169.3, 168.8, 153.7, 153.4, 153.0, 152.2, 151.6, 136.0, 135.4, 129.9, 129.6, 128.4, 125.4, 124.4, 119.8, 118.5, 117.8, 113.5, 112.0, 111.2, 106.5, 98.3, 79.9, 56.2, 54.4, 51.9, 33.6, 30.3, 26.6, 25.6, 21.0, 20.5; LRMS calculated for C₄₆H₄₅N₃O₁₅[M+H]⁺ m/z: 880.3, measured 880.5.

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(butylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate (8h)

¹H NMR (600 MHz, acetone-d6) δ 9.76 (s, 1H), 8.54 (s, 1H), 8.40 (d, J=8.04 Hz, 1H), 7.62 (d, J=1.92 Hz, 1H), 7.47 (d, J=8.04 Hz, 1H), 7.36 (dd, J=8.55 Hz, J=2.07 Hz, 1H), 7.16 (d, J=0.72 Hz, 1H), 6.93-6.88 (m, 2H), 6.86 (d, J=8.58 Hz, 1H), 6.58 (d, J=8.52 Hz, 1H), 6.50-6.46 (m, 2H), 5.78 (s, 4H), 4.20 (s, 4H), 3.83 (s, 3H), 3.19 (t, J=7.05 Hz, 2H), 2.29 (s, 3H), 2.07 (s, 6H), 1.64 (p, J=7.60 Hz, 2H), 1.46 (sext, J=8.28 Hz, 2H), 0.95 (t, J=7.38 Hz, 3H); ¹³C NMR (150 MHz, acetone-d6) δ 170.8, 170.0, 169.3, 168.8, 164.4, 153.7, 153.4, 153.0, 152.2, 138.3, 136.0, 135.4, 134.9, 129.9, 128.4, 125.5, 124.4, 119.8, 118.6, 117.8, 113.5, 111.7, 111.2, 106.5, 97.9, 79.9, 56.2, 54.4, 42.6, 31.9, 30.3, 21.0, 20.9, 20.6, 14.1; LRMS calculated for C₄₄H₄₃N₃O₁₅[M+H]⁺ m/z: 854.3, measured 854.1.

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-3-oxo-6′-(phenylamino)-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate (8i)

¹H NMR (600 MHz, CD₃CN) δ 8.89 (s, 1H), 8.54 (s, 1H), 8.29 (d, J=7.92 Hz, 1H), 7.45 (s, 1H), 7.39 (d, J=7.98 Hz, 1H), 7.35 (t, J=7.83 Hz, 2H), 7.22-7.18 (m, 3H), 7.14 (d, J=2.04 Hz, 1H), 7.06 (t, J=7.35 Hz, 1H), 6.95 (d, J=2.04 Hz, 1H), 6.93 (d, J=8.70 Hz, 1H), 6.88 (dd, J=8.67 Hz, J=2.07 Hz, 1H), 6.83 (d, J=8.58 Hz, 1H), 6.80 (dd, J=8.70 Hz, J=2.01 Hz, 1H), 6.74 (d, J=8.76 Hz, 1H), 5.71 (s, 4H), 4.13 (s, 4H), 3.81 (s, 3H), 2.27 (s, 3H), 2.06 (s, 6H); ¹³C NMR (150 MHz, CD₃CN) δ 170.5, 170.1, 169.0, 164.8, 153.9, 153.0, 152.2, 142.2, 138.3, 135.9, 135.6, 134.5, 130.5, 130.3, 130.2, 128.2, 125.8, 125.3, 123.8, 120.9, 119.9, 117.7, 114.5, 113.8, 111.5, 106.7, 102.0, 80.1, 56.3, 54.4, 21.3, 20.9; LRMS calculated for C₄₆H₃₉N₃O₁₅[M+H]⁺ m/z: 874.2, measured 874.4.

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate (8j)

¹H NMR (600 MHz, CD₃CN) δ 8.90 (s, 1H), 8.57 (s, 1H), 8.29 (dd, J=8.01 Hz, J=1.47 Hz, 1H), 7.45 (d, J=1.98 Hz, 1H), 7.39 (d, J=7.92 Hz, 1H), 7.22-7.18 (m, 2H), 6.96 (d, J=8.70 Hz, 1H), 6.90 (dd, J=8.70 Hz, J=2.16 Hz, 1H), 6.83 (d, J=8.64 Hz, 1H), 6.76 (d, J=9.36 Hz, 1H), 6.34-6.31 (m, 2H), 5.71 (s, 4H), 4.13 (s, 4H), 4.03 (t, J=7.38 Hz, 4H), 3.81 (s, 3H), 2.41 (p, J=7.38 Hz, 2H), 2.27 (s, 3H), 2.06 (s, 6H); ¹³C NMR (150 MHz, CD₃CN) δ 171.1, 170.5, 170.1, 168.7, 164.8, 153.3, 152.2, 138.2, 135.9, 135.2, 134.5, 130.5, 130.3, 128.8, 119.9, 119.4, 113.8, 111.5, 106.7, 97.5, 80.1, 56.3, 54.4, 53.2, 21.3, 20.9, 17.1; LRMS calculated for C₄₃H₃₉N₃O₁₅[M+H]⁺ m/z: 838.2, measured 838.5.

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(3,3-difluoroazetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate (8k)

¹H NMR (600 MHz, CD₃CN) δ 8.88 (s, 1H), 8.51 (s, 1H), 8.27 (dd, J=8.01 Hz, J=1.17 Hz, 1H), 7.45 (d, J=1.80 Hz, 1H), 7.35 (d, J=8.04 Hz, 1H), 7.19 (dd, J=8.58 Hz, J=2.10 Hz, 1H), 7.12 (d, J=2.16 Hz, 1H), 6.88 (d, J=8.64 Hz, 1H), 6.86-6.82 (m, 2H), 6.72 (d, J=8.64 Hz, 1H), 6.44 (d, J=2.28 Hz, 1H), 6.33 (dd, J=8.64 Hz, J=2.34 Hz, 1H), 5.71 (s, 4H), 4.30 (t, J=12.00 Hz, 4H), 4.13 (s, 4H), 3.81 (s, 3H), 2.26 (s, 3H), 2.06 (s, 6H); ¹³C NMR (150 MHz, CD₃CN) δ 171.0, 170.5, 170.1, 169.2, 164.8, 156.2, 153.5, 153.0, 152.7, 152.2, 138.3, 135.9, 135.8, 134.5, 130.1, 130.0, 127.9, 125.2, 124.8, 119.9, 119.0, 117.5, 113.8, 111.5, 110.7, 109.1, 106.6, 100.2, 83.8, 80.1, 64.1, 63.9, 63.8, 56.3, 54.4, 21.2. 20.9; LRMS calculated for C₄₃H₃₇F₂N₃O₁₅[M+H]⁺ m/z: 874.2, measured 874.4.

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-3-oxo-6′-(pyrrolidin-1-yl)-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate (8l)

¹H NMR (600 MHz, CD₃CN) δ 8.92 (s, 1H), 8.53 (s, 1H), 8.27 (dd, J=8.01 Hz, J=1.41 Hz, 1H), 7.45 (dd, J=1.86 Hz, 1H), 7.36 (d, J=7.98 Hz, 1H), 7.20 (dd, J=8.58 Hz, J=2.10 Hz, 1H), 7.14 (d, J=2.16 Hz, 1H), 6.90 (d, J=8.64 Hz, 1H), 6.85 (dd, J=8.67 Hz, J=2.25 Hz, 1H), 6.83 (d, J=8.58 Hz, 1H), 6.68 (d, J=9.42 Hz, 1H), 6.45-6.41 (m, 2H), 5.71 (s, 4H), 4.13 (s, 4H), 3.81 (s, 3H), 3.36-3.34 (m, 4H), 2.27 (s, 3H), 2.06 (s, 6H), 2.03-1.99 (m, 4H); ¹³C NMR (150 MHz, CD₃CN) δ 169.2, 164.9, 153.8, 153.2, 152.2, 138.2, 135.8, 135.4, 134.6, 130.2, 130.0, 128.5, 125.8, 125.2, 119.9, 118.9, 118.0, 113.8, 111.4, 111.3, 106.7, 98.4, 80.1, 56.3, 54.4, 48.7, 41.3, 26.1, 21.3, 20.9; LRMS calculated for C₄₄H₄₁N₃O₁₅[M+H]⁺ m/z: 852.3, measured 852.4.

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-3-oxo-6′-(piperidin-1-yl)-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate (8m)

¹H NMR (600 MHz, acetone-d6) δ 9.76 (s, 1H), 8.55 (s, 1H), 8.40 (d, J=7.86 Hz, 1H), 7.65 (d, J=2.28 Hz, 1H), 7.49-7.46 (m, 1H), 7.38-7.34 (m, 1H), 7.16 (d, J=1.98 Hz, 1H), 6.94-6.88 (m, 2H), 6.87-6.84 (m, 1H), 6.80-6.76 (m, 2H), 6.71-6.67 (m, 1H), 5.78 (app t, 4H), 4.19 (s, 4H), 3.82 (s, 3H), 3.32 (s, 4H), 2.29 (app t, 3H), 2.07 (app t, 6H), 1.69-1.60 (m, 6H); ¹³C NMR (150 MHz, acetone-d6) δ 170.8, 169.9, 169.3, 168.8, 164.3, 156.1, 154.6, 153.4, 153.2, 152.9, 152.2, 138.4, 136.0, 135.6, 134.9, 129.9, 129.4, 128.1, 125.3, 124.4, 119.8, 118.6, 117.6, 113.5, 113.3, 111.2, 108.1, 106.5, 106.4, 102.1, 79.9, 56.2, 54.4, 49.7, 30.3, 26.1, 25.0, 21.0, 20.6; LRMS calculated for C₄₅H₄₃N₃O₁₅[M+H]⁺ m/z: 866.3, measured 866.4.

Example 2 Biological Studies

Determination of Photochemical Properties

Evaluation of the photochemical properties of the active Thallium sensing species was accomplished by saponifying 10 μL of each pro-dye stock solution by incubating with 90 μL of 0.1 M KOH for 20 min at 37° C. with the exception of 8b which was treated with 1% NH₄OH for 1 h at 37° C. The saponified compounds were diluted into 150 mM KCl buffered with 10 mM HEPES, pH=7.22 with 50 μM of EDTA. All photochemical measurements were taken in this buffer. Absorbance and fluorescence emission spectra were recorded on a Molecular Devices Spectromax M5 using polystyrene cuvettes with path length=1 cm at 23° C. Molar absorptivities are reported for λ_max. The emission wavelengths monitored for recording excitation spectra, the excitation wavelengths used for recording emission spectra, and the corresponding emission cutoff filters used are listed in Table 2 below, and representative excitation and emission spectra are shown in FIG. 7 . Spectra for 8b and 8i were not recorded because they exhibit very weak fluorescence.

	TABLE 2

	Excitation Spectra	Emission Spectra

	Wavelength	Cutoff	Wavelength	Cutoff
Compounds	(nm)	(nm)	(nm)	(nm)

8a	535	530	480	495
8g, 8h, 8k	540	530	505	515
8c-f, 8j, 8l, 8m	560	550	520	530

Cell Culture

HEK-293 cells co-expressing GIRK1 and GIRK2 were cultured to 80% confluence in a T75 flask (Techno Plastic Products, Trasadingen, CH) at 37° C. and 5% CO₂in medium consisting of Minimal Essential Medium (Cellgro, Manassas, Va.) containing 1× Glutagro (Cellgro, Manassas, Va.) and 10% (v/v) heat-inactivated fetal bovine serum (Life Technologies, Carlsbad, Calif.). The cells were dislodged by treatment with TrypLE (Gibco/Life Technologies, Carlsbad, Calif.), diluted in medium, and plated on clear bottomed, black-walled, amine coated 384 well plates (BD Biosciences, Billerica, Mass.) at a density ˜20,000 cells per well. The plated cells were incubated overnight at 37° C. and 5% CO₂and used for the assay the next day.

Fluorescence Microscopy

Images were obtained using a Molecular Devices ImageXpress Micro XL. GIRK1 and 2 co-expressing HEK-293 cells were plated in 384-well, clear bottom, black-walled, amine coated plates as described above. Culture medium was replaced by 20 μL of assay buffer (1× HBSS, 4 mM NaHCO₃, and 20 mM HEPES, pH=7.3) containing either 1 μM of Thallos, 5 μM of 8c, or 1 μM of rhodamine 123 (Sigma-Aldrich, St. Louis, Mo.). Cells were incubated in the dark for 1 h at room temperature. The dye loading buffer was then replaced with assay buffer with 1 μg/mL of Hoechst 33342 (Life Technologies, Carlsbad, Calif.) and incubated for 15 min at room temperature. The second dye loading buffer was then replaced with 20 μL of assay buffer supplemented with 1 mM of Allura Red AC (Sigma-Aldrich, St. Louis, Mo.). The cells were then imaged with the ImageXpress Micro XL using the 40× objective and FITC filters for Thallos and Rhodamine 123, Cy3 filters for 8c, and DAPI filters for Hoechst 33342. Representative results obtained by this procedure are shown in FIGS. 2A-2C.

In alternative experiments, HEK cells co-expressing GIRK 1 and GIRK2 were imaged by confocal microscopy using a Zeiss LSM880 AiryScan and a 40×/1.30 C Plan-Apochromat Oil objective on an 8-well IbiTreat μ-Slide (Ibidi, Madison, Wis.). Thallos was imaged using a 488 nm laser and emission collected 493-576 nm, 8c and Rhodamine 123 were imaged using a 514 nm laser and emission collected 519-683 nm, and Hoechst was imaged using a 405 nm laser and emission collected 462-680 nm. Cells were plated on the slide the previous day and culture medium was replaced by 20 μL of assay buffer (1×HBSS, 4 mM NaHCO₃, and 20 mM HEPES, pH=7.3) containing either 1 μM of Thallos, 5 μM of 8c, or 1 μM of rhodamine 123 (Sigma-Aldrich, St. Louis, Mo.). Cells were incubated in the dark for 1 h at room temperature. The dye loading buffer was then replaced with assay buffer with 1 μg/mL of Hoechst 33342 (Life Technologies, Carlsbad, Calif.) and incubated for 15 min at room temperature. The second dye loading buffer was then replaced with 20 μL of assay buffer and the cells were imaged. Representative results obtained by this procedure are shown in FIGS. 2D-2F.

Flux Assays—General Procedure

Dye loading—Compounds 8a-m were diluted to a concentration of 5 M in assay buffer (1×HBSS, 4 mM NaHCO₃, and 20 mM HEPES, pH=7.3). Dye loading solutions of Thallos were prepared by dissolving a 25 μg aliquot in 30 μL of 6.7% Pluoronic F-127 (w/v) in DMSO and diluting the resulting solution 1:1000 into assay buffer. Culture medium was removed and replaced with 20 μL of dye loading buffer and incubated in the dark at room temperature for 1 h.

Assay—Assays were conducted on a WaveFront Biosciences Panoptic 1 or 2 (WaveFront Biosciences, Franklin, Tenn.) as indicated. Dye loading solutions were removed and replaced with assay buffer immediately before imaging unless otherwise noted. 10 s following commencement of plate imaging, 20 μL of compound at 2.5× concentration in assay buffer was added and incubated for 120 s at which point 10 μL of Tl⁺ at 5× concentration in stimulus buffer (125 mM NaHCO₃, 1.8 mM CaSO₄, 1 mM MgSO₄, 5 mM glucose, 10 mM HEPES, pH 7.4, and T12SO₄), and images taken for the following 120 s.

On/off ratio of 8a-m determined in HEK-293 cells co-expressing GIRK1 and GIRK2—The general Tl⁺ flux assay procedure outlined above was conducted on a WaveFront Biosciences Panoptic 1 using a checkerboard of 10 μM of ML297 (final concentration) and vehicle. 1 mM of T12SO₄(final concentration) was used for stimulus. Assay and stimulus buffers were supplemented with 2 mM of Allura Red AC. Compounds 8c-m were imaged using 517/20 nm excitation and 562/40 nm emission filters and 8a and 8b were imaged using 480/40 nm excitation and 538/40 nm emission filters (Semrock, Rochester, N.Y.). On/off ratios were calculated by dividing the fluorescence intensity after saturation following Tl⁺ addition by the fluorescence intensity immediately after compound addition (n=6).

ML297 concentration response curves—In general, all concentration-response data were collected on a WaveFront Biosciences Panoptic 2. Thallos treated cells were imaged using 482/35 nm excitation and 536/40 nm emission filters. 8c treated cells were imaged using 529/24 nm excitation and 565/24 nm emission filters (unless otherwise noted). FluxOR Red treated cells were imaged using 544/24 nm excitation and 593/40 nm emission filters (all filters purchased from Semrock, Rochester, N.Y.). FluxOR Red reagent was prepared according to the manufacturer's procedure. 5 μM of 8c was used for dye loading for all conditions. Dye loading solutions of Thallos were prepared by following the reagent preparation steps listed above and diluting this 1:1000 into the corresponding dye loading buffer.

FluxOR Red procedure with background suppressor. The manufacturer's procedure was followed with the exceptions that the dye loading solution was replaced with 20 μL of assay buffer, solutions of ML297 were prepared in assay buffer plus background suppressor, 20 μL of compound solution was added and 10 μL stimulus buffer (basal) was added according to the general assay protocol described above.

FluxOR Red procedure without background suppressor. The FluxOR Red manufacturer's procedure was followed except the background suppressor was replaced by H₂O in all buffers.

Bicarbonate buffer with Allura Red AC. The assay and stimulus buffers described in the general Tl⁺ flux procedure were used for dye loading, assay buffer with ML297, and stimulus buffer. All buffers were supplemented with 1 mM of Allura Red AC. The wash step to remove dye loading buffer was omitted.

Bicarbonate buffer without Allura Red AC. The general Tl⁺ flux procedure was followed without any solutions supplemented with Allura Red AC.

Data from all experiments were processed as follows. The fluorescence intensity values of the first 6 time points for each well were averaged and the fluorescence intensity for all time points in the well was divided by this value to provide F/F_ofor each time point. The average F/F_oof the vehicle control was then subtracted from each curve and the normalized, control subtracted fluorescence intensity value was sampled at a time point several seconds after Tl⁺ addition. These data were averaged (n=6-12 for each concentration of ML297) and fit to a curve using GraphPad Prism. Fold increases in fluorescence reported in the main paper were calculated by averaging F/F_obetween 100 and 110 s after Tl⁺ addition. Data presented in FIG. 3 and FIG. 6 were obtained using 8c with the FluxOR Red kit as described above under the FluxOR Red with background suppressor procedure.

pKa Determination

Saponified 8c and Thallos were prepared and stored as 10 mM solutions in DMSO. These were diluted to 200 μM in DMSO. 5 μL of these solutions were diluted into 195 μL of buffers consisting of 150 mM KCl, 50 μM EDTA, and 10 mM of either potassium citrate (pH=4-6), potassium phosphate (pH=6.25-8), or Tris-HCl (pH=8-9.5) on a 96-well, black walled, clear bottom plate (Greiner Bio-one, Monroe, N.C.). Fluorescence measurements were recorded on a Biotek Neo Synergy plate reader using excitation and emission wavelengths of 485 nm and 528 nm for Thallos and 500 nm and 565 nm for 8c. Fluorescence intensity data were average for each pH (n=3) and the data fit to a curve using GraphPad Prism.

Results

To determine the in vitro photochemical properties of the active Tl⁺ sensing species, 8a-m were treated with base to remove the acetoxymethyl esters and aryl acetate and then diluted into 10 mM HEPES buffered 150 mM KCl with 50 μM EDTA at pH=7.22. Saponified 8a-m exhibited photochemical properties comparable to previously reported rhodols (Table 1).

The performance of rhodol Tl⁺ sensors in the Tl⁺ flux assay was assessed by incubating HEK-293 cells co-expressing G-protein-gated inwardly rectifying K⁺ (GIRK) channel subunits GIRK1 and GIRK2 with 8a-m in dye loading buffer for 1 hour prior to addition of 1 mM (final concentration) of Tl⁺ stimulus and the fold increase in brightness calculated. Dimethyl rhodol 8c demonstrated a 4.21-fold increase in brightness in the presence of Tl⁺ in HEK-293 cells. A notable decrease in ability to sense Tl⁺ among the dialkyl series 8c-8f as the alkyl chain length increases was observed. Monoalkyl rhodols 8g and 8h as well as cyclic amine containing rhodols 8j, 8l, and 8m exhibit >3 fold increases in brightness upon addition of Tl⁺ stimulus. Gem-difluoro 8k exhibited almost half the response to Tl⁺ compared with the non-fluorinated analog 8j. Phenyl rhodol 8i exhibited very low initial fluorescence in HEK-293 cells requiring a longer exposure time than the other dyes and a minor increase in fluorescence upon addition of Tl⁺. Fluorescence was not detected in HEK-293 cells incubated with acetamide 8b before or after the addition of Tl⁺. Considering photochemical properties and response to Tl⁺ in HEK-293 cells, dimethyl rhodol 8c was identified as the best of the series and further evaluated for its utility as a reagent for Tl⁺ flux assays.

Cytoplasmic localization of Tl⁺ indicators is desirable for Tl⁺ flux assays of ion channels and transporters localized to the plasma membrane. The cellular localization of 8c was compared to Thallos by treating GIRK1 and GIRK2 co-expressing HEK-293 cells with Thallos, 8c, or the mitochondrial stain Rhodamine 123 for 1 h and all counterstained with the nuclear stain Hoechst. Cells were then imaged by fluorescence microscopy. 8c exhibited diffuse cytoplasmic staining comparable to Thallos, in contrast to the punctuate mitochondrial staining observed with Rhodamine 123 (FIGS. 2A-F). These results demonstrate that 8c localizes predominantly to the cytoplasm.

The pK_as of 8c and Thallos were determined to be 5.23 and 6.52, respectively (FIG. 4 ) indicating that 8c may be superior to Thallos when conducting assays at the lower range of physiological pH.

The performance of 8c was evaluated in a Tl⁺ flux assay to generate a concentration response curve (CRC) of the GIRK activator ML297. HEK-293 cells co-expressing GIRK1 and GIRK2 were loaded with 5 μM of 8c for 1 h, treated with 0.01-10 μM of ML297, and then subjected to Tl⁺ stimulus (1 mM final concentration). 8c exhibited a 3.09±0.004-fold increase in fluorescence at the highest ML297 concentration (FIG. 3A). Normalized, control subtracted fluorescence intensity values were sampled along the curves following Tl⁺ addition at each concentration and a CRC was fit using GraphPad Prism (FIG. 3B). The EC₅₀was determined to be 257±3.7 nM while the same procedure utilizing Thallos produced an EC₅₀of 272±23 nM, which was not statistically significantly different.

Using the same cell line and GIRK activator, 8c was also compared to the commercially available, red-shifted Tl⁺ sensor FluxOR Red (Molecular Probes). Following the manufacturer's procedure, which requires supplementation of assay and stimulus buffers with a proprietary background suppressor, FluxOR Red exhibited a 5.37±0.001-fold increase in fluorescence at the highest concentration of ML297 compared to Thallos (4.30±0.008-fold increase) and 8c (3.09±0.004-fold increase). However, omission of the FluxOR Red background suppressor resulted in a significantly diminished increase in fluorescence of 1.17±0.007 at the highest ML297 concentration for FluxOR Red while Thallos and 8c only showed modest decreases in the absence of background suppressor (2.80±0.009 and 2.87±0.004-fold increases in fluorescence, respectively, FIG. 5 ). In addition, 8c exhibited a comparable dynamic range and EC₅₀results using 544/24 nm excitation and 593/40 nm emission filters indicating 8c can also be used very effectively with longer wavelength, off-peak filters (FIGS. 6A-6B).

Strict adherence to the FluxOR Red protocol may not be compatible with or convenient for screening certain channels or transporters. For example, many buffers for Tl⁺ flux assays, including those in most commercial kits, utilize gluconate as the primary anion due to the insolubility of Tl⁺ in Cl⁻ containing buffers. However, gluconate-based buffers may not be optimal for all targets and assay systems. In addition, the components of extracellular fluorescence masking dyes may affect the pharmacology of targets of interest. Tolerance of the Tl⁺ sensing dye towards varied assay conditions would allow greater flexibility for screening. The performance of 8c, Thallos, and FluxOR Red were compared using a bicarbonate-based stimulus buffer with 500 μM of Tl⁺ under two conditions. First, an automation compatible “no-wash” procedure was tested where the wash step after dye loading was omitted and all buffers were supplemented with 1 mM of Allura Red AC to suppress extracellular fluorescence. Under these conditions, 8c and Thallos exhibited 3.40±0.009 and 3.83±0.007-fold increases in fluorescence and ML297 EC₅₀s of 204±5.2 nM and 219±23 nM, respectively, were calculated. In contrast, FluxOR Red exhibited no change in fluorescence upon Tl⁺ addition and the data could not be fit to a CRC. Second, using similar conditions omitting Allura Red AC while employing a wash step after dye loading produced comparable results (FIG. 5 and Table 3). These data demonstrate the utility of 8c in assays where extracellular fluorescence masking dyes are not compatible or where “no wash” procedures are desirable.

TABLE 3

pEC₅₀and standard error of ML297 activation of GIRK1 and GIRK2
co-expressing HEK-293 cells determined from concentration response
curves fit using data obtained under the corresponding conditions.

	FluxOR Red Kit	HCO₃ ⁻ Stimulus Buffer
	Background Suppressor
	1 mM Allura Red AC

Dye	+	−	+	−

Thallos	6.57 ± 0.04	6.65 ± 0.02	6.66 ± 0.01	6.61 ± 0.01
FluxOR Red	6.64 ± 0.01	6.41 ± 0.03	ND	7.02 ± 0.1
8c	6.59 ± 0.01	6.61 ± 0.02	6.69 ± 0.01	6.59 ± 0.03
8c (544x/	6.63 ± 0.01	6.66 ± 0.04	—	—
593m)

In conclusion, dimethyl rhodol 8c has been identified from a series of rhodol-based Tl⁺ sensors as a versatile reagent for Tl⁺ flux assays. It localizes to the cytoplasm similarly to Thallos while exhibiting greater pH tolerance and excitation and emission spectra red-shifted ˜30 nm. 8c exhibits an excellent dynamic range using both on-peak and longer off-peak filter sets and is compatible with a variety of assay conditions.

The foregoing discussion discloses and describes merely exemplary embodiments of the invention. One skilled in the art will readily recognize from such discussion and from the accompanying drawings and claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims.

Claims

We claim:

1. A compound of the following formula,

or a salt thereof,

wherein:

Z is O, CR^xR^y, SiR^xR^y, or SO₂;

R¹is C_1-10alkyl-C(O)—;

—NR²R³is —NHC(O)CH₃, —N(CH₃)₂, —N(CH₂CH₃)₂, —N(CH₂CH₂CH₃)₂, —N(CH₂CH₂CH₂CH₃)₂, —NH-cyclohexyl, —NH—CH₂CH₂CH₂CH₃, —NH-phenyl, azetidin-1-yl, 3,3-difluoroazetidin-1-yl, pyrrolidin-1-yl, or piperidin-1-yl;

R⁴is C_1-10alkyl;

R⁵and R⁶are each independently C_1-6alkyl, —C_1-4-alkylene-O—R^z, —C_1-4-alkylene-O—C(O)—R^z, or —C_1-4-alkylene-O—C(O)—O—R^z;

R^xand R^yare each independently C_1-4alkylene; and

R^z, at each occurrence, is independently C_1-6alkyl, C_3-10cycloalkyl, C_6-20aryl, or a 4 to 8-membered heterocycle, wherein the C_3-10cycloalkyl, C_6-20aryl, and 4 to 8-membered heterocycle are optionally substituted with one or more substituents independently selected from the group consisting of halogen, C_1-4haloalkyl, —CN, C_1-4alkyl-O—, C_1-4alkyl-C(O)—NH—, and C_1-4alkyl-NH—C(O)—.

2. The compound of claim 1, or a salt thereof, wherein Z is O.

3. The compound of claim 1, or a salt thereof, wherein R¹is C_1-4alkyl-C(O)—.

4. The compound of claim 1, or a salt thereof, wherein R⁴is C_1-4alkyl.

5. The compound of claim 1, or a salt thereof, wherein —NR²R³is —N(CH₃)₂.

6. The compound of claim 1, or a salt thereof, wherein R⁵and R⁶are independently —C_1-4-alkylene-O—C(O)—R^z, and each R^zis independently C_1-6alkyl.

7. The compound of claim 1, or a salt thereof, wherein each of R⁵and R⁶is —CH₂—O—C(O)—CH₃.

8. The compound of claim 1, having a structure of formula (I-a), or a salt thereof,

wherein —NR²R³, R⁵, and R⁶are as defined in claim 1.

9. The compound of claim 1, selected from the group consisting of

bis(acetoxymethyl) 2,2′-((4-(3′-acetamido-6′-acetoxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxamido)-2-methoxyphenyl)azanediyl)diacetate;

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(diethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dipropylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dibutylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;

bis(acetoxymethyl) 2,2′((4-(3′-acetoxy-6′-(cyclohexylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;

bis(acetoxymethyl) 2,2′((4-(3′-acetoxy-6′-(butylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-3-oxo-6′-(phenylamino)-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;

bis(acetoxymethyl) 2,2′((4-(3′-acetoxy-6′-(3,3-difluoroazetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-3-oxo-6′-(pyrrolidin-1-yl)-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate; and

bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-3-oxo-6′-(piperidin-1-yl)-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate,

or a salt thereof.

10. The compound of claim 9 that is bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxamido)-2-methoxyphenyl)azanediyl)diacetate, or a salt thereof.

11. A method of preparing the compound of claim 1, or a salt thereof, the method comprising reacting a compound of the following formula

with a compound of the following formula

to provide the compound of claim 1, or salt thereof.

12. The method of claim 11, further comprising converting a compound of the following formula

to the compound of formula

13. The method of claim 11, wherein Z is O.

14. A method of detecting flux of a metal ion into a cell, the method comprising

(a) contacting the cell with a compound of the following formula:

or a salt thereof,

wherein:

Z is O;

R¹is C_1-10alkyl-C(O)—;

—NR²R³is —N(CH₃)₂, —N(CH₂CH₃)₂, —N(CH₂CH₂CH₃)₂, —N(CH₂CH₂CH₂CH₃)₂, —NH-cyclohexyl, —NH—CH₂CH₂CH₂CH₃, azetidin-1-yl,3,3-difluoroazetidin-1-yl, pyrrolidin-1-yl, or piperidin-1-yl;

R⁴is C_1-10alkyl;

R⁵and R⁶are each independently C_1-6alkyl, —C_1-4-alkylene-O—R^z, —C_1-4-alkylene-O—C(O)—R^z, or —C_1-4-alkylene-O—C(O)—O—R^z; and

R^z, at each occurrence, is independently C_1-6alkyl, C_3-10cycloalkyl, C_6-20aryl, or a 4 to 8-membered heterocycle, wherein the C_3-10cycloalkyl, C_6-20aryl, and 4 to 8-membered heterocycle are optionally substituted with one or more substituents independently selected from the group consisting of halogen, C_1-4haloalkyl, —CN, C_1-4alkyl-O—, C_1-4alkyl-C(O)—NH—, and C_1-4alkyl-NH—C(O)—;

(b) subsequently contacting the cell with the metal ion; and

(c) subsequently measuring fluorescence produced by the cell.

15. The method of claim 14, wherein the metal ion is Tl⁺.

16. The method of claim 14, wherein the cell comprises at least one ion channel.

17. The method of claim 16 wherein the at least one ion channel comprises a potassium channel.

18. The method of claim 14, further comprising measuring fluorescence produced by the cell after step (a) and before step (b).